The long range objective of this research is to develop an understanding, at the molecular level, of the mechanisms of regulation of transcription by RNA polymerase III in Xenopus laevis. The differential expression of the somatic and oocyte-type 5S RNA genes during the early development of Xenopus is a model system for investigating how patterns of gene expression are established and maintained during cellular differentiation. Although this system has been the focus of intense investigation and is one of the best understood systems of eukaryotic gene control, it continues to provide surprises. We have recently found that differential transcription of the somatic and oocyte-type 5S RNA genes in a whole oocyte extract is a consequence of different rates of stable transcription complex assembly on the different genes. These findings identify a general molecular mechanism for differential gene expression which may provide the key distinguishing feature for the developmental control of the families of 5S RNA genes in Xenopus and which may also be utilized in other systems of regulated gene expression. The design of this research proposal includes a detailed study of the kinetics of transcription complex formation on a variety of class III genes, coupled with an investigation of the protein-DNA interactions involved. In addition to 5S RNA and tRNA genes, we propose to clone and study the 7SL and U6 genes from Xenopus laevis. Although these genes are transcribed by RNA polymerase III, their distinct promoter structures suggest different control mechanisms may be used. Microinjection of specific clones into Xenopus embryos will be used to correlate in vitro transcription results with the in vivo environment. Fractionation of the whole oocyte extract will be used to identify the component of the transcription system which displays differential interaction kinetics. We are also developing a novel procedure to separate active transcription complexes from inactive templates in order to characterize the DNA-protein interactions on actively transcribed genes and to examine alterations in these interactions which occur during the passage of the RNA polymerase through the genes. Through these studies we hope to develop an understanding of the integration of the molecular processes involved in the control of expression of the variety of class III genes.